Microbiology (2006), 152, 1819–1826
DOI 10.1099/mic.0.28522-0
Staphylococcus aureus adheres to human
intestinal mucus but can be displaced by certain
lactic acid bacteria
Satu Vesterlund,1 Matti Karp,2 Seppo Salminen1 and Arthur C. Ouwehand1,3
1
Functional Foods Forum, Department of Biochemistry and Food Chemistry, University of
Turku, Turku, Finland
Correspondence
Satu Vesterlund
2
[email protected]
Department of Environmental Engineering and Biotechnology, Tampere University of
Technology, Tampere, Finland
3
Danisco Innovations, Kantvik, Finland
Received 14 September 2005
Revised
3 January 2006
Accepted 10 January 2006
There is increasing evidence that Staphylococcus aureus may colonize the intestinal tract,
especially among hospitalized patients. As Staph. aureus has been found to be associated with
certain gastrointestinal diseases, it has become important to study whether this bacterium can
colonize the intestinal tract and if so, whether it is possible to prevent colonization. Adhesion is the
first step in colonization; this study shows that Staph. aureus adheres to mucus from resected
human intestinal tissue. Certain lactic acid bacteria (LAB), mainly commercial probiotics, were
able to reduce adhesion and viability of adherent Staph. aureus. In displacement assays the amount
of adherent Staph. aureus in human intestinal mucus was reduced 39–44 % by Lactobacillus
rhamnosus GG, Lactococcus lactis subsp. lactis and Propionibacterium freudenreichii subsp.
shermanii. Moreover, adherent Lactobacillus reuteri, Lc. lactis and P. freudenreichii reduced
viability of adherent Staph. aureus by 27–36 %, depending on the strain, after 2 h incubation.
This was probably due to the production of organic acids and hydrogen peroxide and possibly
in the case of L. reuteri to the production of reuterin. This study shows for the first time that Staph.
aureus can adhere to human intestinal mucus and adherent bacteria can be displaced and
killed by certain LAB strains via in situ production of antimicrobial substances.
INTRODUCTION
Staphylococcus aureus is an opportunistic pathogen causing
a broad range of nosocomial and community-acquired
infections. Diseases caused by this bacterium can range from
skin infections to foodborne illnesses and severe infections
such as endocarditis, osteomyelitis and sepsis (Lowy, 1998).
The nasal carriage of Staph. aureus is common, 20–50 % of
the population (Cespedes et al., 2005), but also intestinal
carriage appears to be increased among hospitalized patients
(Dupeyron et al., 2001; Ray et al., 2003; Rimland & Roberson,
1986; Squier et al., 2002) and infants (Lindberg et al., 2000).
Lindberg et al. (2000) showed that over 75 % of Swedish
infants have Staph. aureus in their stools while Bjorksten
et al. (2001) showed that 65 % of infants have these bacteria
in their stools. Towards adulthood the intestinal carriage of
Staph. aureus decreases due to increased complexity of the
adult microbiota and so-called ‘colonization resistance’,
meaning that the indigenous intestinal microbiota provides
protection against colonization of the gastrointestinal tract
Abbreviation: LAB, lactic acid bacteria.
0002-8522 G 2006 SGM
by exogenous micro-organisms (Lindberg et al., 2004; van
der Waaij et al., 1971).
As the microbiota covering the intestinal epithelium has
a protective role in preventing colonization of ingested
bacteria, certain bacterial strains belonging to the healthy
intestinal microbiota can be isolated and used as probiotics.
Probiotics are ‘live micro-organisms which when administered in adequate amounts confer a health benefit on the
host’ (WHO, 2001). There are several reports showing that
specific probiotic strains protect against gastrointestinal
infections (Gorbach et al., 1987; Saavedra et al., 1994;
Vanderhoof et al., 1999). Different mechanisms for this have
been suggested, such as overall reduction of the gut pH, a
direct antagonism against pathogens (production of antimicrobial components such as hydrogen peroxide and
bacteriocins), competition for the same binding sites as
pathogens, stimulation of the immune system and competition for nutrients (Collins & Gibson, 1999).
The aim of the present study was to assess whether Staph.
aureus can adhere to healthy human colonic mucus and
whether adhesion and viability of potentially adherent
Staph. aureus can be reduced by specific lactic acid bacteria;
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1819
S. Vesterlund and others
a preliminary investigation was made of the possible
mechanisms for such effects.
METHODS
used led to a linear relationship between added and bound bacteria
and thus constant adhesion percentages. Moreover, the saturation
level, when the numbers of added bacteria are too high, leading to
underestimation of the percentage bound bacteria, was not reached.
Human intestinal mucus. Resected human intestinal tissue was
Bacterial strains and growth conditions. The Staphylococcus
aureus strains used were RN4220, which is derived from the strain
8325-4 (Kreiswirth et al., 1983), and a bioluminescent variant of the
same strain, Staph. aureus RN4220/pAT19 (Vesterlund et al., 2004).
Salmonella enterica serovar Typhimurium ATCC 14028 was used as
a negative control in adhesion assays, as in previous experiments it
has exhibited low adhesion (Vesterlund et al., 2005). The 11 strains
of lactic acid bacteria (LAB) used are listed in Table 1. All bacterial
stocks were stored at 286 uC in 40 % (v/v) glycerol. Staph. aureus
and Sal. enterica serovar Typhimurium were plated first and subsequently cultured by inoculating one colony into Luria–Bertani
broth (LB; yeast extract and tryptone were from Pronadisa). When
adhesion was measured, 10 ml ml21 of [59-3H]thymidine (16?7 Ci
mmol21; 618 GBq mmol21) was added to the cultures to metabolically radiolabel the bacteria. In the case of the bioluminescent
Staph. aureus strain, broth and plates were supplemented with 10 mg
erythromycin ml21. Staph. aureus and Sal. enterica serovar Typhimurium cultures were grown for 16 h without agitation at 30 uC to
reach stationary growth phase. LAB strains were grown in de Man,
Rogosa and Sharpe (MRS) broth (Oxoid) and they were inoculated
directly as a 0?5 % inoculum from the glycerol stocks. When adhesion kinetics was measured, the LAB cultures were supplemented
with 10 ml [59-3H]thymidine ml21. In the case of Lb. reuteri 40 mM
glycerol was added as a substrate for production of reuterin in the
culture broth (Talarico et al., 1988). LAB were grown in anaerobic
conditions for 20 h at 37 uC (except for Lc. lactis subsp. lactis and P.
freudenreichii subsp. shermanii JS, which were grown for 2 days at
30 uC) in order to reach the late exponential growth phase. All
bacterial strains were harvested by centrifugation and washed twice
with 1 ml phosphate-buffered saline (PBS; pH 7?2). The OD600 of
the bacterial suspensions was adjusted with PBS to 0?5±0?02,
corresponding to 0?56108 c.f.u. ml21 for Lb. acidophilus La5, 1–
26108 c.f.u. ml21 for Lb. casei Shirota, Lb. johnsonii LA1, Lb. rhamnosus GG and Lc. lactis subsp. lactis and 2–46108 c.f.u. ml21 for the
remaining strains. Although the number of added bacteria varied
from 0?56108 to 46108 c.f.u. ml21, the bacterial concentrations
used as a source of mucus. The use of resected tissue was approved
by the joint ethical committee of the University of Turku and the
Turku University Central Hospital and informed written consent
was obtained from the patient. The tissue sample used in this study
was from ascending colon and obtained from a colorectal cancer
patient from the healthy area adjacent to the tumour. The intestinal
material was processed as described earlier (Ouwehand et al., 2002).
In short, resected material was collected on ice within 20 min and
processed immediately by washing gently with PBS containing
0?01 % gelatin. Mucus was collected by gently scraping with a rubber
spatula into a small amount of HEPES-Hanks buffer (10 mmol
HEPES l21; pH 7?4) and centrifuged (13 000 g, 10 min). After measurement of the protein content, the mucus was stored at 220 uC.
In adhesion assays the mucus was diluted to a protein concentration
of 0?5 mg ml21 with HEPES-Hanks buffer. Mucus was passively
immobilized on a polystyrene microtitre plate (Maxisorp, Nunc;
and in bioluminescence measurements B&W Isoplate 1450-581,
PerkinElmer) as a volume of 100 ml by incubating overnight at 4 uC
(Ouwehand et al., 2003).
Adhesion assay. After overnight incubation the mucus-coated
microtitre plate wells were washed three times with 250 ml HEPES-
Hanks buffer. Then radiolabelled Staph. aureus bacteria were added
to the wells in a volume of 100 ml (in competition assays in a volume
of 50 ml, i.e. 50 ml of Staph. aureus incubated alone or together with
50 ml of LAB). Four parallel wells were used in each experiment.
Bacteria were allowed to adhere for 1 h at 37 uC and the wells were
washed three times with 250 ml HEPES-Hanks buffer to remove the
nonadherent bacteria. In exclusion assays LAB were incubated first
with the mucus, then washed away and followed by incubation with
radiolabelled Staph. aureus. Similarly in displacement assays radiolabelled Staph. aureus was incubated first with the mucus, then
washed away and followed by incubation with LAB. The bacteria
bound to mucus were released and lysed with 1 % SDS/0?1 M
NaOH by incubation at 60 uC, followed by measurement of radioactivity by liquid scintillation. Sal. enterica serovar Typhimurium was
Table 1. Lactic acid bacteria used in the study
Bacterium
Lactobacillus acidophilus La5*
Lactobacillus casei ShirotaD
Lactobacillus johnsonii LA1D
Lactobacillus paracasei-33d
Lactobacillus plantarumD
Lactobacillus reuteri ING1d
Lactobacillus rhamnosus GGD
Lactococcus lactis subsp. lactisD
Enterococcus faeciumD
E. faecium SF68d
Propionibacterium freudenreichii
subsp. shermanii JSD
Source
Chr. Hansen, Hørsholm, Denmark
Yakult, Tokyo, Japan
Nestlé, Lausanne, Switzerland
Uni-President Enterprises Corp., Tainan Hsien, Taiwan
American Type Culture Collection (ATCC 8014)
Ingmanfoods, Söderkulla, Finland
Valio, Helsinki, Finland
Valio, Helsinki, Finland
Arla Foods, Viby, Denmark
Oriola, Espoo, Finland
Valio, Helsinki, Finland
*A generous gift from Dr B. Grenov, Christian Hansen A/S, Hørsholm, Denmark.
DA generous gift from Dr M. Saxelin, Valio Ltd, Helsinki, Finland.
dIsolated from a commercial product containing the strain.
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Microbiology 152
Staph. aureus adherence to human intestinal mucus
used as a negative control in adhesion assays. The adhesion ratio
(%) of bacteria was calculated by comparing the radioactivity of the
adhered bacteria to that of the added bacteria.
Viability of adherent bacteria. The bioluminescent indicator
strain has been used earlier in the screening of antimicrobial substances produced by LAB against Staph. aureus (Vesterlund et al.,
2004). In short, this indicator strain allowed stable light production
since it harboured luxAB genes responsible for light production as
well as luxCDE genes responsible for the production of the substrate
(long-chain fatty aldehyde) for the reaction. The effect of adherent
LAB on the viability of adherent Staph. aureus was determined in a
competition assay. This ensured that the number of adherent Staph.
aureus was the same regardless of the presence or absence of LAB.
After adhesion and washings, the wells were covered either with
HEPES-Hanks or with LB supplemented with 1 % glucose. HEPESHanks was used as it is used in adhesion assays, whereas LB supplemented with glucose allows the effect of available nutrients on
viability to be observed. Results were calculated after 2 h incubation
by comparing the viability of the sample to the viability of the
adherent Staph. aureus incubated without LAB.
Antimicrobial substances produced by LAB. The production of
antimicrobial substances by those strains which were able to reduce
viability of Staph. aureus was studied. A newly developed assay was
used for this purpose (Vesterlund et al., 2004). This assay allows
detection of organic acids, hydrogen peroxide or bacteriocins produced by LAB. In short, LAB were grown as described above and the
culture supernatants were collected by centrifugation, filter-sterilized
(0?22 mm pore size) and supplemented with erythromycin. Erythromycin was used as the used indicator strain carries an erythromycin
resistance marker. When the production of hydrogen peroxide and
bacteriocins was determined, the supernatants were neutralized to
pH 7?2 with 4 M NaOH and phosphate buffer (pH 7?2; 0?1 M
phosphate final concentration). To determine possible production of
hydrogen peroxide by LAB, the supernatants were treated with catalase; to determine possible effects of bacteriocins, the supernatants
were treated with proteinase K (both enzymes were purchased from
Sigma and used at a concentration of 1 mg ml21). MRS was used as
a negative control and nisin (10 IU ml21) as a positive control in
the assay and they were treated in a similar way as supernatants.
Determination of maximum number of adhered bacteria on
mucus and dissociation constants of bacteria
Theory. Michaelis–Menten-type dissociation kinetic models have
been used to describe adhesion kinetics of bacteria (Lee et al., 2000).
Briefly, the equation:
Assay. The adhesion assay was performed with twofold dilution
series from each bacterium and followed the protocol described
above.
Statistical analysis. Pair-wise Student’s t-test was used to deter-
mine the significance (P<0?05) of differences between the control
and the samples. Results shown are from three or four independent
experiments.
RESULTS
Adhesion of bacteria to human intestinal mucus
and effect of LAB on adhesion ability of Staph.
aureus
Among the tested LAB, three strains showed relatively high
adhesion: for Lb. rhamsosus GG, Lc. lactis subsp. lactis and
P. freudenreichii subsp. shermanii JS the adhesion ratios
were 11?5 %, 10?1 % and 11?3 %, respectively (Table 2).
Staph. aureus showed similar adhesion as Lb. acidophilus
La5, 4?4 % and 4?0 %, respectively (Table 2), and showed
higher adhesion (P<0?05) than the rest of the tested strains.
Three of the LAB strains, Lb. casei Shirota, Lb. paracasei-33
and E. faecium adhered poorly, expressing similar adhesion
as the negative Salmonella control (0?4 %).
When the effect of LAB on the adhesion ability of Staph.
aureus was tested in displacement, exclusion and competition
assays, statistically significant effects (P<0?05) of certain
LAB were seen only in the displacement assay. Interestingly,
the same strains that expressed high adhesion ratios were
also able to displace Staph. aureus from mucus: Lb. rhamnosus GG reduced the amount of adherent Staph. aureus by
44 %, Lc. lactis by 41 % and P. freudenreichii by 39 % after
1 h (Table 3). Furthermore, a trend of reduced adhesion of
Staph. aureus was seen with Lb. rhamnosus in competition
Table 2. Adhesion (%) of bacteria to human intestinal
mucus
Results shown are means±SD of three independent experiments (in
the case of Staph. aureus, mean±SD of six independent experiments).
Bacterium
Adhesion (%)
kz1
Bacterial cell (x)zmucus (e) / ? Bacteriummucus complex (ex )
k{1
is in equilibrium. When the concentration of free bacterial cells is
(x2ex), the dissociation constant (kd) for the process can be written
as kd=(k21)/(k+1)= (x2ex)x/ex. Rearrangement of the equation
gives the concentration of the bacterium–mucus complex: ex=e?x/
(kd+x). When x is very much larger than kd, ex approaches e. As a
result the maximum value of ex is obtained when mucus is saturated
with bacteria as em, which may be written as ex=em?x/(kd+x). This
equation can be further rearranged to give a linear relationship:
1=ex ~1=em zkd =ðem .xÞ
ð1Þ
Hence, plots of 1/ex against 1/x give straight lines, in which the
intercepts on the ordinate give the values of 1/em and those on the
abscissa give the values of 21/kd.
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Lb. acidophilus La5
Lb. casei Shirota
Lb. johnsonii LA1
Lb. paracasei-33
Lb. plantarum
Lb. reuteri ING1
Lb. rhamnosus GG
Lc. lactis subsp. lactis
E. faecium
E. faecium SF68
P. freudenreichii subsp. shermanii JS
Staph. aureus RN4220
Sal. enterica serovar Typhimurium
(negative control)
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4?0±1?5
0?4±0?1
2?6±0?6
0?5±0?1
1?7±0?7
1?1±0?2
11?5±3?1
10?1±2?4
0?3±0?1
1?1±0?1
11?3±3?6
4?4±1?2
0?4±0?1
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S. Vesterlund and others
Table 3. Effect of LAB on adhesion ability of Staph. aureus
The results (means±SD of four independent experiments) are represented as percentages compared to
adhesion of Staph. aureus without LAB (taken as 100 %).
LAB
Lb. acidophilus La5
Lb. casei Shirota
Lb. johnsonii LA1
Lb. paracasei-33
Lb. plantarum
Lb. reuteri ING1
Lb. rhamnosus GG
Lc. lactis subsp. lactis
E. faecium
E. faecium SF68
P. freudenreichii subsp. shermanii JS
Displacement
Exclusion
Competition
74?0±22?7
85?2±16?3
69?7±13?2
73?5±21?7
72?8±12?5
81?1±22?0
56?1±9?7*
59?2±13?6*
74?9±24?3
78?5±36?7
60?7±13?5*
126?6±35?6
110?8±30?7
109?9±33?4
112?1±14?5
111?4±23?1
109?0±32?7
97?0±16?9
86?3±25?8
103?0±23?0
85?9±31?0
79?0±26?0
96?7±47?4
87?9±21?1
91?2±35?1
96?5±20?1
82?4±35?4
93?5±18?9
84?9±14?0
80?4±36?4
71?5±19?9
62?9±17?0
94?7±28?6
*These LAB significantly reduce adhesion of Staph. aureus (P<0?05).
(15 %; P=0?24), with Lc. lactis in exclusion (14 %; P=0?21)
and in competition (20 %; P=0?32), and with P. freudenreichii in exclusion (21 %; P=0?23) after 1 h, but statistical
significance was not reached due to relatively high variation
between experiments.
Maximum number of adhered bacteria on
mucus and dissociation constants of bacteria
When the reciprocal concentrations of adhered bacteria were
plotted against the reciprocal concentrations of the added
bacteria, in all cases a linear relationship was observed. With
two tested bacteria, Lb. casei Shirota (See Fig. 1) and P.
freudenreichii subsp. shermanii JS, two linear regions were
observed. This may mean that two binding mechanisms are
involved for these bacteria, one for a high bacterial concentration (lower affinity and thus probably non-specific
adhesion when the adhesion sites are masked due to a high
number of bacteria; forces such as van der Waals and
hydrophobic interactions included) and one for a lower
Fig. 1. Adhesion kinetics of Lb. casei Shirota. The lines indicate the linear fit according to the least-squares method. &,
Low bacterial concentration; X, high bacterial concentration.
1822
concentration, which implies higher affinity and thus probably specific adhesion. Thus at low bacterial concentration,
the adhesion of bacterial cells on mucosa involves the
maximum number of adhesion sites, and at high bacterial
concentration there is self-competition for the adhesion and
thus minimum numbers of adhesion sites are involved (Lee
et al., 2000).
The linear relationships of the most adhesive strains, Lb.
rhamnosus, Lc. lactis, P. freudenreichii and Staph. aureus, are
shown in Fig. 2. By using equation 1, the maximum number
of adhered bacteria on mucus (em) and dissociation constants (kd) for each strain were calculated, and are summarized in Table 4. The values were calculated as c.f.u. per
well, which compares a mucus area of 0?1 cm2. As shown in
Table 4, among the 12 bacterial strains tested, Lb. plantarum
Fig. 2. Double-reciprocal representation of the adhesion of Lb.
rhamnosus, Lc. lactis, P. freudenreichii and Staph. aureus to
human intestinal mucus. The lines indicate the linear fit according to the least-squares method. &, Lb. rhamnosus; X, Lc.
lactis; m, P. freudenreichii; 6, Staph. aureus.
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Microbiology 152
Staph. aureus adherence to human intestinal mucus
Table 4. Maximum number of adhered bacteria (em) on human intestinal mucus and dissociation constant (kd) of bacteria
Results shown are means±SD of three or four independent experiments. Low bacterial concentration:
number of added bacteria <7?76106 c.f.u. per well for Lb. casei and <5?66105 c.f.u. per well for P.
freudenreichii.
Bacterium
Lb. acidophilus La5
Lb. casei Shirota
High concn
Low concn
Lb. johnsonii LA1
Lb. paracasei-33
Lb. plantarum
Lb. reuteri ING1
Lb. rhamnosus GG
Lc. lactis subsp. lactis
E. faecium
E. faecium SF68
P. freudenreichii subsp. shermanii JS
High concn
Low concn
Staph. aureus RN4220
Maximum no. of
adhered bacteria (em)
(c.f.u. per well)
Dissociation
constant, kd
(c.f.u. per well)
2?06105D
5?56106d
3?76105d
1?86104d
3?66105D
8?46104D
1?46107
1?26106D
3?76105D
6?76106*
2?76105D
3?76105D
3?06108*
1?26106d
8?36106d
5?16107
1?26109
3?86108
1?26106d
5?16107D
8?16107
2?26107D
3?16106
1?56105D
3?36106
2?46107D
6?36105d
7?76107
*Significantly higher compared to Staph. aureus (P<0?05).
DSignificantly lower compared to Staph. aureus (P<0?05).
dSignificantly lower compared to Staph. aureus (P<0?001).
had the highest amount of adhered bacteria on mucus
(1?46107 c.f.u. per well) and the em was 170 times higher
compared to Lb. paracasei-33, which had the lowest em
(8?46104 c.f.u. per well). However, Lb. plantarum showed
an adhesion ratio of only 1?7 % (Table 2); this is likely to be
due to its having the highest kd among the tested strains,
1?26109 c.f.u. per well (Table 5), implying low affinity for
adhesion to mucus. Staph. aureus had the third highest em
among the tested strains and this also explained its relatively
high adhesion ability (4?4 %; Table 2). The em for Staph.
aureus was 3?36106 c.f.u. per well and only Lb. plantarum
(1?46107 c.f.u. per well) and Lc. lactis subsp. lactis
(6?76106 c.f.u. per well) had a higher em. However, the
kd of Staph. aureus was relatively high, 7?76107 c.f.u. per
well, indicating that Staph. aureus dissociates from mucus
more easily compared to six tested LAB: Lb. acidophilus
La5 (5?56106 c.f.u. per well; P<0?001), Lb. johnsonii
LJ1 (8?36106 c.f.u. per well; P<0?001), Lb. rhamnosus
(1?26106 c.f.u. per well; P<0?001), Lc. lactis (5?16107
c.f.u. per well; P<0?05), E. faecium SF68 (2?26107 c.f.u. per
well; P<0?05) and P. freudenreichii (2?46107 c.f.u. per well;
P<0?05). With lower bacterial concentrations, Lb. casei
Shirota and P. freudenreichii also showed tighter binding
(P<0?001) to mucus compared to Staph. aureus: 1?26106
c.f.u. per well, 6?36105 c.f.u. per well and 7?76107 c.f.u.
per well, respectively. However, these lower bacterial
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Table 5. Effect of adherent LAB on the viability of adherent
Staph. aureus after 2 h
The results (means±SD of three independent experiments) are
represented as percentages compared to viability of adherent
Staph. aureus without LAB (taken as 100 %).
LAB
Lb. acidophilus La5
Lb. casei Shirota
Lb. johnsonii LA1
Lb. paracasei-33
Lb. plantarum
Lb. reuteri ING1
Lb. rhamnosus GG
Lc. lactis subsp. lactis
E. faecium
E. faecium SF68
P. freudenreichii subsp.
shermanii JS
HEPES-Hanks
LB with 1 %
glucose
93?0±1?3*
106?1±9?0
100?8±8?9
101?2±7?5
98?4±4?5
98?3±4?7
105?8±7?7
97?8±8?7
97?0±5?4
98?2±10?3
99?6±7?3
98?1±13?3
106?9±8?7
87?1±7?8
111?9±5?2
85?4±15?4
72?7±10?0*
76?9±14?6
64?4±3?6*
89?2±8?1
86?9±7?8
72?7±7?6*
*These LAB significantly reduce viability of Staph. aureus (P<0?05).
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S. Vesterlund and others
concentrations were not used in displacement, exclusion
and competition assays (Table 3).
Effect of adherent LAB on the viability of
adherent Staph. aureus
The effect of adherent LAB on the viability of adherent
Staph. aureus was determined by using a sensitive reporter
system based on a bioluminescent Staph. aureus indicator
strain. We have previously shown that bioluminescence
emission correlates closely with the viability of Staph. aureus
(Vesterlund et al., 2004). This has been proven also with
several other bacterial strains (Beard et al., 2002; Rocchetta
et al., 2001; Unge et al., 1999). When bacteria were allowed
to adhere to the mucus, the non-bound bacteria were
washed away and the microtitre plate wells were covered
with HEPES-Hanks, Lb. acidophilus La5 was able to reduce
the viability of Staph. aureus (Table 5). However, when the
wells were filled with culture medium, Lb. acidophilus had
no effect on Staph. aureus. Although most of the strains were
able to reduce viability of Staph. aureus in the presence of
culture medium, a statistically significant reduction (27–
36 %; P<0?05) was obtained with Lb. reuteri, Lc. lactis and
P. freudenreichii.
Antimicrobial substances produced by LAB
Supernatants of Lb. reuteri, Lc. lactis and P. freudenreichii
were collected, neutralized and treated with catalase or
proteinase K. Proteinase K treatment did not cause recovery
of bioluminescence when compared to non-proteinasetreated but neutralized supernatant, indicating that LAB
were not producing bacteriocins against Staph. aureus.
However, either catalase treatment or neutralization caused
recovery, indicating that hydrogen peroxide and organic
acids had antimicrobial activity against Staph. aureus.
DISCUSSION
The number of both community-acquired and hospitalacquired staphylococcal infections has increased steadily
(Kielian et al., 2001). Treatment of these infections has
become difficult due to emergence of antibiotic-resistant
strains, and new agents to treat and especially prevent
staphylococcal infections are needed. The possible intestinal
carriage of Staph. aureus may have negative health effects;
for example during antibiotic treatment it can lead to the
overgrowth of bacteria in the intestine and thus antibioticassociated diarrhoea (Ackermann et al., 2005; Boyce &
Havill, 2005). Also association of Staph. aureus with inflammatory bowel disease has been suggested, as lumen-derived
Staph. aureus superantigens have been shown to elicit
inflammation in a mouse model (Lu et al., 2003). Moreover,
there is accumulating evidence that the colon may serve as a
reservoir of antibiotic-resistance genes (Salyers et al., 2004),
for example vancomycin-resistant Staph. aureus (VRSA)
(Ray et al., 2003). Although it is unclear whether Staph.
aureus belongs to the normal human colon microbiota, it
seems that at least among hospitalized patients colonization
1824
is possible (Donskey, 2004). The hypothesis of intestinal
colonization is also supported by a recent study showing
that the caecal mucus layer may provide an important niche
for intestinal colonization by Staph. aureus (Gries et al.,
2005).
As Staph. aureus has been found to adhere to nasal mucin
(Shuter et al., 1996), we hypothesized here that adhesion to
intestinal mucus, of which the main components are mucins,
would be possible as well. Moreover, in earlier studies
several bacteria have been found to adhere to intestinal
mucin oligosaccharides (Moncada et al., 2003). In the
present study we used a model based on human intestinal
mucus obtained from resected colonic tissue to assess
whether Staph. aureus adheres to mucus. Human cell-lines
Caco-2 and HT-29 do not produce mucus and the mucusproducing cell line HT-29-MTX (Lesuffleur et al., 1990)
produces mainly mucins with gastric immunoreactivity
(MUC3 and MUC5C) and only few mucins with colonic
immunoreactivity (MUC2 and MUC4) (Lesuffleur et al.,
1993). Intestinal epithelial cells offer an important model for
studying adhesion of bacteria to intestinal areas without a
mucus layer, such as Peyer’s patches, or areas where the
mucus is eroded due to disease, but they can not be used as
models for adhesion to mucus. Another advantage of the
use of mucus is that the colon’s own mucosa-associated
microbiota is present and its effect on adhesion is also taken
into account. A drawback is the availability of the mucus and
also the need to process it immediately.
Here we show for the first time that Staph. aureus can adhere
to human colonic mucus but can be displaced by specific
LAB. Lb. rhamnosus GG, Lc. lactis subsp. lactis and P.
freudenreichii subsp. shermanii were able to displace Staph.
aureus from human colonic mucus by 39–44 %. Interestingly, the displacement capability was restricted to the LAB
with relatively high adhesion ability, Lb. rhamnosus GG, Lc.
lactis subsp. lactis and P. freudenreichii subsp. shermanii JS,
with adhesion ratios of 11?5 %, 10?1 % and 11?3 %, respectively (Table 2). Mathematical modelling including determination of the maximum number of adhered bacteria on
mucus (em) and the binding affinity (kd) to mucus as well as
measurement of viability of adherent Staph. aureus were
used to explain the mechanism of displacement. Staph.
aureus showed the third highest em among the tested
bacteria. Only Lb. plantarum and Lc. lactis had higher em
values. This also explained the relatively high binding of
Staph. aureus to mucus. However, the binding affinity of
Staph. aureus to mucus was only moderate (7?76107 c.f.u.
per well; Table 4), and the highest affinity to mucus was
obtained with Lb. rhamosus GG (1?26106 c.f.u. per well).
This indicates that Staph. aureus can be outcompeted by
probiotics which have higher affinity to the mucus. This
is likely to explain why Lb. rhamnosus, Lc. lactis and P.
freudenreichii displaced Staph. aureus from mucus. Similarly
under in vivo conditions, Staph. aureus would probably be
washed out more easily from the intestinal mucus surface
than for example Lb. rhamnosus GG. However, in competition
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Microbiology 152
Staph. aureus adherence to human intestinal mucus
assays, LAB showing higher affinity than Staph. aureus to
mucus were not able to reduce its adhesion. This may have
been due to the amounts of bacteria used: in displacement
the adherent pathogens were covered with LAB and outnumbered whereas in competition the amounts of bacteria
were similar. In exclusion assays there was no effect of LAB
on adhesion of Staph. aureus, indicating that the bacteria do
not use same adhesion receptors.
When viability of adherent Staph. aureus was measured in
the presence of adherent LAB, the LAB had an effect only
when nutrients were available. Adherent Lb. reuteri, Lc. lactis
and P. freudenreichii significantly reduced the viability of
Staph. aureus by 27–36 % within 2 h. The reduction of
viability was not due to competition for nutrients (which
were present in excess) but rather to the in situ production of
organic acids and hydrogen peroxide, and in the case of
Lb. reuteri possibly reuterin (Arques et al., 2004; Vesterlund
et al., 2004). Uehara et al. (2001) showed that colonization
of meticillin-resistant Staph. aureus (MRSA) in the oral
cavities of newborns was inhibited by the viridans group of
streptococci, and that this was probably due to the production of hydrogen peroxide by these streptococci.
However, it is unclear whether LAB can produce antimicrobial substances against Staph. aureus in vivo. It is also
possible that the hydrogen peroxide produced is degraded
by the metabolism of other bacteria (Ryan & Kleinberg,
1995).
The emergence of antibiotic resistance among Staph. aureus
strains and possibly increased intestinal colonization of these
bacteria require alternative methods for prevention and
treatment of staphylococcal diseases. Our results show that
Staph. aureus adheres to human colonic mucus and that
certain LAB could have antiadhesive and antimicrobial
effects against this bacterium. However, it remains for
further studies to show that other virulent Staph. aureus
strains can adhere to colonic mucus in vitro and in vivo,
and to show that LAB have antiadhesive and antimicrobial
effects against Staph. aureus also in vivo.
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ACKNOWLEDGEMENTS
Financial support was obtained from the Academy of Finland (grant
number 53758), the Danisco Foundation and the Finnish Food
Research Foundation.
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